Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are set forth at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Fungi are eukaryotic microorganisms that are universally distributed. In nature, fungi play a major role in the decomposition of plant materials. They are also responsible for spoilage of food and in the preparation of beverages and pharmaceuticals. Out of an estimated 100,000 species of fungi described by mycologists, approximately 150 species are recognized as pathogens in humans and animals. The increasing incidence of AIDS and the development of newer treatments for hematologic malignancies and solid organ transplants has led to an increase in the number of immunocompromised patients. These patients have a high risk of developing fungal infections, which, if not rapidly diagnosed and treated, are capable of causing death in a matter of days. The number of antifungal drugs is limited and their toxic side effects on the patient are much higher than that of comparable antibacterial therapy. Therefore, a rapid diagnosis of a fungal infection with administration of appropriate therapy is critical in these patients.
Aspergillus species, for example, are associated with allergic bronchopulmonary disease, mycotic keratitis, otomycosis, nasal sinusitis, and invasive infection. The most severe disease caused by the aspergilli occurs in immunocompromised patients with invasive pulmonary infection followed by rapid dissemination. The frequency of invasive aspergillosis (IA), as well as other invasive mold infections has increased.in recent years due to the expanding number of patients receiving aggressive chemotherapy regimens and immunosuppressive agents (2). The nonspecific symptoms and the lack of rapid diagnostic assays to detect these infections have been major problems in treating patients with invasive disease Early recognition of invasive fungal infection and treatment with appropriate antifungal therapy is key to reducing the mortality associated with disseminated disease (25). The mortality rate for bone marrow transplant patients with pulmonary IA is greater than 70% (5, 15). Due to the typically long time required for identification of a mold using standard culture procedures, most patients with suspected disease are treated empirically with amphotericin B (AmB). Resistance to AmB as well as itraconazole has been reported for some Aspergillus species although the number of isolates studied in each case is limited (14, 16).
Unfortunately, the identification of the aspergilli based on morphologic methods requires adequate growth for evaluation of colony characteristics and microscopic features. A culture time of 5 days or more is generally required for identification of anamorphic forms of Aspergillus. There are more than 180 species in the Aspergillus genus, although three, including A. flavus, A. fumigatus and A. terreus account for the vast majority of IA infections. A. nidulans, A. niger and A. ustus are rarely encountered as causes of invasive disease(18).
Various molecular approaches have been used for the detection of Aspergillus from environmental and clinical samples (3, 6, 27). Targets for the genus-level detection of Aspergillus have included the 18S rRNA gene, mitochondrial DNA, the intergenic spacer region, and the internal transcribed spacer (ITS) regions. The ITS regions are located between the 18S and 28S rRNA genes and offer distinct advantages over other molecular targets including increased sensitivity due to the existence of approximately 100 copies per genome. The rRNA gene for 5.8S RNA separates the two ITS regions. The sequence variation of ITS regions has led to their use in phylogenetic studies of many different organisms (9, 26).
It would be highly advantageous if means were available to differentiate and efficiently identify clinically relevant pathogenic fungi. It is an object of the present invention to provide compositions, methods and kits to accomplish this goal.